A pilot study of idiotypic vaccination for follicular B-cell lymphoma using a genetic approach. CRC NO: 92/33. Protocol NO: PH1/027.
Explore the source record for details and available documents.
Antibodies, Anti-Idiotypic↗
Biomedical subjects
S J Russell
Publications and source records attributed to S J Russell.
ABC of clinical haematology. The future of haematology, molecular biology, and gene therapy.
Explore the source record for details and available documents.
Anemia↗
A gene delivery system activatable by disease-associated matrix metalloproteinases.
We are developing protease-activatable gene delivery vehicles for selective gene delivery to protease-expressing cells. Angiogenesis, inflammation, and cancer invasion are linked to the overexpression of matrix metalloproteinases (MMPs), which destroy the extracellular matrix. Therefore, the MMPs are promising targets for therapy. We have displayed epidermal growth factor (EGF) on retroviral vector particles as an MMP-cleavable amino-terminal extension of the 4070A murine leukemia virus (MLV) envelope glycoprotein. This was achieved by engineering an MMP-cleavage signal (PLGLWA) into the linker between the EGF domain and the 4070A SU. The chimeric envelope was expressed and incorporated into viral particles, and the EGF domain could be cleaved from the surface of the viral particles by gelatinase A (MMP-2). The MMP-sensitive vector and control MMP-insensitive vectors could bind, via their displayed EGF domains, to EGF receptors on A431 cells but were unable to infect them because the EGF receptor (EGFR) does not support postbinding steps required for retroviral entry. In the presence of exogenous MMPs, the infectivity of the MMP-sensitive vector, but not of the MMP-insensitive vectors, was restored on A431 cells, and this cleavage activation could be partially blocked by MMP inhibitors. Endogenous MMPs produced by EGFR-positive HT 1080 cells could selectively activate the MMP-sensitive vector giving rise to a titer that was 1,000-fold higher on HT 1080 cells than on MMP-negative A431 cells. Inhibitor studies and gelatin zymograms indicated that the membrane-associated MT-MMP expressed on the HT 1080 cells played an important role in cleavage activation of the vector. When presented simultaneously with both EGFR-positive cell lines A431 and HT 1080, the vector could efficiently discriminate between the two different cell types, infecting the MMP-positive HT 1080 cells in preference over the A431 cells.
Binding Sites↗
Receptor co-operation in retrovirus entry: recruitment of an auxiliary entry mechanism after retargeted binding.
We have constructed Moloney murine leukemia virus (MoMLV)-derived envelope glycoproteins (AMO) displaying an amino-terminal Ram-1-binding domain in which a variety of different amino acid spacers have been inserted between the displayed domain and the MoMLV surface (SU) subunit. Titres of retroviruses generated with these chimeric envelopes were enhanced on cells expressing both Ram-1 and Rec-1 receptors compared with the titres on cells expressing only one or other receptor type. The absolute viral titres and the degree of titre enhancement due to receptor cooperativity were highly variable between the different chimeric envelopes and were determined primarily by the properties of the interdomain spacer. An extreme example of receptor co-operativity was encountered when testing Ram-1-targeted AMOPRO envelopes with specific proline-rich interdomain spacers. AMOPRO viruses could not enter cells expressing only Rec-1 or only Ram-1 but could efficiently infect cells co-expressing both receptors. The data are consistent with a model for receptor co-operativity in which binding to the targeted (Ram-1) receptor triggers conformational rearrangements of the envelope that lead to complete unmasking of the hidden Rec-1-binding domain, thereby facilitating its interaction with the viral (Rec-1) receptor which leads to optimal fusion triggering.
3T3 Cells↗
Gene therapy for B cell lymphomas.
The use of genes or genetically modified cells for therapeutic benefit is likely to have a significant therapeutic role for patients with B cell lymphomas in the future. To date, most gene therapy strategies applicable to the therapy of these diseases have not reached the point of clinical study. Adoptive immunotherapy using donor leucocyte infusion to treat aggressive B cell neoplasms in immunosuppressed patients has, however, shown great promise clinically, and studies of idiotypic vaccination in patients with low grade B cell neoplasms are also under way. Results from in vitro and animal studies continue to suggest that it may become possible to use the immune system for therapeutic benefit, and many current basic research strategies in the gene therapy of B cell non-Hodgkin's lymphoma are based on immune modulation of T cells or tumour cells themselves. Other major approaches to gene therapy for B cell malignancies include the introduction of directly toxic or "suicide genes" into B cells or the chemoprotection of haemopoietic stem cells by the introduction of drug resistance genes. All of these approaches require efficient and accurate gene transfer as well as correct expression of the gene product within the target cell. Although some way from therapeutic use, specific targeting of gene delivery is an area of active investigation and will be of value in many of the gene therapy strategies applicable to B cell lymphomas.
Cancer Vaccines↗
Isolation and characterization of SUG2. A novel ATPase family component of the yeast 26 S proteasome.
Using a genetic strategy designed to find proteins involved in the function of the Saccharomyces cerevisiae transcriptional activator GAL4, we isolated mutants in two genes which rescue a class of gal4 activation domain mutants. One of these genes, SUG1, encodes a member of a large family of putative ATPases, the Conserved ATPase containing Domain (CAD) proteins (also known as AAA proteins) that are involved in a wide variety of cellular functions. Subsequently, SUG1 was identified as a subunit of the 26 S proteasome. We have now cloned the gene defined by the second complementation group. SUG2 encodes an essential 49-kDa protein that is also a member of the CAD family and is 43% identical to SUG1. The mutation in sug2-1, like that in sug1-1, is found in the CAD near the highly conserved ATPase motif. We present biochemical and genetic evidence that SUG2 is associated in vivo with SUG1 and is a novel CAD protein subunit of the 26 S proteasome. With its highly conserved mammalian homologs, human p42 and ground squirrel CADp44, SUG2 defines a new class of proteasomal CAD proteins.
Adenosine Triphosphatases↗
Retroviral display of antibody fragments; interdomain spacing strongly influences vector infectivity.
Five different single-chain antibody fragments (scFv) against human cell-surface antigens were displayed on murine ecotropic retroviral vectors by fusing them to the Moloney SU envelope glycoprotein. The spacing between the scFv and the SU glycoprotein was varied by fusing the scFv to residue +7 or to residue +1 of Moloney SU and by inserting linker sequences of different lengths between the domains. All of the chimeric envelopes were efficiently incorporated into vector particles and could bind to human cells through their displayed antibody fragments, but did not infect them. The spacing between the scFvs and the SU glycoproteins had no significant effect on the efficiency of envelope expression or viral incorporation and did not affect the binding properties of the chimeric envelopes, nor did it influence the efficiency of targeted gene delivery to human cells by scFv-displaying vectors. However, on murine fibroblasts the infectivity of vectors incorporating the chimeric envelopes was strongly influenced by the length of the interdomain spacer. The titers were very low when the single-chain antibodies were fused through a tripeptide linker to SU residue +7 and were greatly enhanced (up to 10(5)-fold) when they were fused to SU residue +1 through a heptapeptide linker. These results point to the importance of steric interactions between the domains of chimeric envelope glycoproteins and may have implications for retroviral vector design for human gene therapy.
Amino Acid Sequence↗
Vectors for cancer gene therapy.
Many viral and non-viral vector systems have now been developed for gene therapy applications. In this article, the pros and cons of these vector systems are discussed in relation to the different cancer gene therapy strategies. The protocols used in cancer gene therapy can be broadly divided into six categories including gene transfer to explanted cells for use as cell-based cancer vaccines; gene transfer to a small number of tumour cells in situ to achieve a vaccine effect; gene transfer to vascular endothelial cells (VECs) lining the blood vessels of the tumour to interfere with tumour angiogenesis; gene transfer to T lymphocytes to enhance their antitumour effector capability; gene transfer to haemopoietic stem cells (HSCs) to enhance their resistance to cytotoxic drugs and gene transfer to a large number of tumour cells in situ to achieve nonimmune tumour reduction with or without bystander effect. Each of the six strategies makes unique demands on the vector system and these are discussed with reference to currently available vectors. Aspects of vector biology that are in need of further development are discussed in some detail. The final section points to the potential use of replicating viruses as delivery vehicles for efficient in vivo gene transfer to disseminated cancers.
Genetic Therapy↗
Vaccine strategies in non-Hodgkin's lymphomas.
As described above, the most recent advances in anti-idiotype vaccination strategies have gone hand in hand with recent developments in molecular biology and other forms of cancer therapy. The techniques that are currently available in antibody engineering will greatly facilitate protein production and purification and will reduce the time and effort needed to produce the patient specific vaccines. Cytokine (gene) therapy has extensively been studied in cancer treatment and cancer vaccination and some therapeutic strategies are currently being evaluated in clinical trials (Bubenik, 1996). Combination therapy of idiotypic vaccination with cytokine therapy has recently been explored with promising results. The main focus so far has been on GM-CSF and IL-2, although other cytokines might prove to be efficient in stimulating different effector arms of the immune system. The nature of the immune response mounted by the host against the tumour and the mechanisms by which the tumour cells escape the effector functions of the immune system are not yet fully known. A better knowledge of the nature of B-cell lymphomas and the relation to the patient's immune system will therefore benefit the further development of the therapeutic strategies. Further research will provide us with a better view of how to break the immune tolerance and of which components of the immune system have to be targeted in order to obtain optimal therapeutic results.
Cancer Vaccines↗
Peptide-displaying phages for targeted gene delivery?
Explore the source record for details and available documents.
Bacteriophages↗
Improvement of retroviral retargeting by using amino acid spacers between an additional binding domain and the N terminus of Moloney murine leukemia virus SU.
We previously reported a strategy to redirect the retroviral host range by expressing single-chain antibodies (S. J. Russell, R. E. Hawkins, and G. Winter, Nucleic Acids Res. 21:1081-1085, 1993) or ligands (F.-L. Cosset, F. J Morling, Y. Takeuchi, R. A. Weiss, M. K. L. Collins, and S. J. Russell, J. Virol. 69:6314-6322, 1995) at the N terminus of Moloney murine leukemia virus (MoMLV) surface proteins (SU). Although such chimeric envelopes were able to bind the new receptors, the transduction efficiency of retargeted viruses was generally low. We hypothesized that conformational rearrangements of envelope glycoproteins were not optimally triggered following binding, and to overcome these postbinding blocks, we have generated here a set of chimeric MoMLV-derived envelopes targeted to the Ram-1 phosphate transporter in which we have varied the spacing between the Ram-1-binding domain and the MoMLV SU. All of the recombinant envelopes were correctly expressed on virions, and all bound efficiently to Ram-1. However, the interdomain spacing greatly affected the efficiency of gene transfer by retroviral vectors that had bound to Ram-1 via their chimeric envelopes. Optimal interdomain spacing allowed a 100-fold-increased viral transduction via Ram-1 compared to our previous results.
3T3 Cells↗
Targeting of retroviral vectors through protease-substrate interactions.
Targetable, injectable vectors would greatly facilitate the development of in vivo therapy strategies. Viral and nonviral vectors can be targeted through ligand-receptor interactions, but protease-substrate interactions have not previously been exploited for vector targeting. Epidermal growth factor (EGF) was fused to a retroviral envelope glycoprotein via a cleavable linker comprising a factor Xa protease recognition signal. Vector particles displaying the cleavable EGF domain could bind to EGF receptors on human cells but did not transfer their genes until they were cleaved by factor Xa protease, whereupon gene delivery proceeded normally. Proteolytic activation of receptor-targeted vectors can therefore provide the basis for a novel two-step targeting strategy that may facilitate efficient targeted in vivo delivery of therapeutic genes.
3T3 Cells↗
Allogeneic bone marrow transplantation for children with acute lymphoblastic leukemia conditioned with busulfan, cyclophosphamide and melphalan.
Children with acute lymphoblastic leukemia (ALL) undergoing allogeneic bone marrow transplantation (BMT) using total body irradiation (TBI)-containing conditioning regimens are at risk of substantial late sequelae affecting growth and endocrine functions. This has lead to the use of non-TBI or chemotherapy conditioning regimens for ALL patients in many centers. However, it is unknown whether chemotherapy conditioning results in improved antileukemic efficacy or reduced transplant-related toxicity. We report our experience using a chemotherapy conditioning regimen (busulfan (Bu), cyclophosphamide (CY) and melphalan (Mel)) in 26 consecutively enrolled children with ALL (group I). At a median follow-up of 58 months, event-free survival (EFS) for Bu/CY/Mel-treated patients was 27% (+/- 8.7), while the leukemic relapse rate was 49% (+/- 13). Regimen-related toxicity was severe in these patients: overall treatment-related mortality was 42%, while 50% of patients had interstitial pneumonitis and 35% of patients had severe hemorrhagic cystitis. A historical control group of 25 consecutively enrolled patients, mostly treated with TBI-containing regimens (group II), had an EFS of 36% (+/- 9.6%) and a leukaemic relapse rate of 45% (+/- 11) at a median follow-up of 117 months. We conclude that the Bu/CY/Mel conditioning regimen leads to severe transplant-related toxicity and did not significantly improve leukemic relapse rates.
Adolescent↗
Targeting retrovirus entry.
Most of the clinical gene therapy trials that have been initiated to date have employed ex vivo strategies in which cells are genetically modified outside the body and reimplanted. The ability to deliver genes accurately and efficiently to selected target cell populations in vivo would greatly expand the scope of gene therapy, but current vectors are not well suited to this task. Here we review recent attempts to develop retroviral vectors incorporating engineered envelope glycoproteins that are capable of delivering their genes in a highly specific manner to selected human target cells.
Amino Acid Sequence↗
Tissue-specific gene expression from Mo-MLV retroviral vectors with hybrid LTRs containing the murine tyrosinase enhancer/promoter.
Transcriptional tissue specificity was engineered directly into Moloney Murine Leukaemia Virus (Mo-MLV)-derived retroviral vectors by replacing the viral enhancer in the 3' long terminal repeat (LTR) with two different lengths (2.5 kbp or 769 bp) of the murine tyrosinase promoter/enhancer. The hybrid tyrosinase-LTR was transferred to the proviral 5' LTR following viral packaging and infection of target cell lines. Hybrid tyrosinase-LTR-driven IL-2 production was barely above background levels in infected nonmelanoma cell lines containing intact provirus, whereas infected melanoma cell lines expressed high levels of IL-2 and the larger tyrosinase promoter/enhancer fragment directed higher levels of transgene expression. By replacing the viral enhancer with the tyrosine promoter/enhancer sequences, promoter interference effects which we have previously observed when the tyrosinase promoter was included as an internal promoter within a similar retroviral vector were effectively abolished. Our data show that the hybrid tyrosinase-LTR behaves as a tightly regulated melanocytic-specific regulatory element when embedded in an enhancer-deleted Mo-MLV LTR. The use of other heterologous cellular promoter/enhancer elements in similar vectors should allow the development of simpler, targeted retroviral vectors for the expression of genes in selected cell types and may eventually provide for the development of safer, more efficient vectors for use in human gene therapy.
3T3 Cells↗
p53 mutations, O6-alkylguanine DNA alkyltransferase activity, and sensitivity to procarbazine in human brain tumors.
BACKGROUND: In human brain tumors, sensitivity to procarbazine as measured by sensitivity in a xenograft tumor model correlated inversely with amounts of the DNA repair enzyme O6-alkylguanine DNA alkyltransferase (AT). METHODS: To test the hypothesis that mutations of the p53 tumor suppressor gene in human tumors also can correlate with the response to chemotherapy, p53 mutations2 were identified in primary human malignant brain tumors and cell lines in which AT activity and procarbazine sensitivity in a xenograft model was ascertained. RESULTS: Mutations were identified in 7 of 21 (33%) specimens tested. Specimens containing p53 mutations tended to exhibit an increased growth delay in procarbazine-treated xenografts and lower amounts of AT. CONCLUSIONS: p53 mutations in brain tumors may contribute to procarbazine sensitivity by failing to induce arrest at the G1/S cell-cycle checkpoint, thereby preventing the repair of procarbazine-induced genetic alterations.
Animals↗
Retroviruses as vectors.
Recombinant retroviruses have long been used to deliver heterologous genes to mammalian cells. Convenient packaging cell lines and vector plasmids have been distributed widely and 'home-made' retroviral vectors have now become a useful research tool in many laboratories. Compared to more traditional methods of gene transfer, retroviral vectors are extraordinarily efficient gene delivery vehicles which cause no detectable harm as they enter their target cells. In the nucleus the retroviral necleic acid becomes integrated into chromosomal DNA, ensuring its long-term persistence and stable transmission to all future progeny of the transduced cell. Up to 8 kilobases of foreign gene sequence can be packaged in a retroviral vector and this is more than enough for most gene therapy applications. Retroviral vectors can also be manufactured in large quantities to meet very stringent safety specifications. They have therefore been selected as the vectors of choice in 80% of the clinical gene therapy trials that have been approved to date. So far there have been no reported short- or long-term toxicity problems associated with their use in human gene therapy trials, now dating back to 1989. However, despite this impressive record, there is still great scope (and need) for the development of new, improved retroviral vectors and packaging systems to fuel further advances in the field of human gene therapy. In the following discussion, existing retroviral vectors are reviewed and current areas of technological development are emphasised.
Gene Expression Regulation, Viral↗
Random X chromosome inactivation in a female with a variant of Wiskott-Aldrich syndrome.
A 15-month-old female presented with eczema, thrombocytopenia, recurrent infections and failure to thrive. She had low serum IgM and IgG subclasses and an abnormal lymphocyte proliferative response to periodate in vitro. Molecular X chromosome inactivation analysis, using the polymorphic HUMARA DNA probe, showed that the infant has random X chromosome inactivation. We conclude that she has an atypical form of Wiskott-Aldrich syndrome which may be inherited in an autosomal recessive manner.
Cell Division↗